Transpedicular injection of rhBMP-2 with β-tricalcium phosphate to reduce the proximal junctional kyphosis after adult spinal deformity correction: preliminary study

The aim of this preliminary study was to assess the impact of injecting recombinant human bone morphogenetic protein-2 (rhBMP-2) with β-tricalcium phosphate (β-TCP) carrier into the uppermost instrumented vertebra (UIV) during surgery to prevent the development of proximal junctional kyphosis (PJK) and proximal junctional failure (PJF). The 25 patients from study group had received 0.5 mg rhBMP-2 mixed with 1.5 g β-TCP paste injection into the UIV during surgery. The control group consisted of 75 patients who underwent surgery immediately before the start of the study. The incidences of PJK and PJF were analyzed as primary outcomes. Spinopelvic parameters and patient-reported outcomes were analyzed as secondary outcomes. Hounsfield unit (HU) measurements were performed to confirm the effect of rhBMP-2 with β-TCP on bone formation at preoperative and postoperative at computed tomography. PJK and PJF was more occurred in control group than study group (p = 0.02, 0.29, respectively). The HU of the UIV significantly increased 6 months after surgery. And the increment at the UIV was also significantly greater than that at the UIV-1 6 months after surgery. Injection of rhBMP-2 with β-TCP into the UIV reduced PJK and PJF rates 6 months after surgery with new bone formation.


Intraoperative local administration of rhBMP-2 into the UIV
Pedicle screw diameter and length were anticipated using preoperative computed tomography (CT).Screws with a diameter and length 2 and 5 mm smaller, respectively, than the largest possible screw from the CT scan, were inserted at the UIV level.For example, if the CT scan showed that a 6.5 mm × 45 mm screw could be inserted into the UIV, a 4.5 mm × 40 mm screw was inserted.After screw placement at all vertebral levels was completed, an intraoperative CT scan with O-arm® (Medtronic, Minneapolis, MN, USA) was performed.Screw trajectory was then confirmed from the O-arm® image, and the screws at the UIV level were removed to make the prepathway of rhBMP-2 to vertebral body.Bone wax was applied at the entry hole of the previously inserted screws to block blood oozing from the trabecular bone, followed by rhBMP-2 injection.A mixture of 0.5 mg rhBMP-2 and 1.5 g β-TCP carrier (CGbio, Seoul, Korea) (Fig. 1) with 0.2 ml normal saline was carefully injected through the bone wax and into the screw hole, immediately followed by insertion of pedicle screws of definitive diameter and length.Total amount of injection was less than 0.5 cc at respective one pedicle screw hole.There was no visible leakage outside of bone wax.

Radiographic analysis of PJK/F and sign of new bone formation
Spinopelvic parameters including pelvic incidence (PI), LL, PI-LL, SVA, thoracic kyphosis (TK), and the amounts of surgical correction were measured using biplanar stereo radiographic full-body imaging (EOS, Paris, France) 13 .
Figure 1.β-tricalcium phosphate which is used as carrier for rhBMP-2 at this study.
PJK was defined as (1) postoperative proximal junctional sagittal Cobb angle > 15° and (2) change in the proximal junctional sagittal Cobb angle from the preoperative measurement of > 15°.The proximal junction is between the upper endplate of the vertebra two-level superjacent to the UIV and the lower endplate of the UIV (Fig. 2).PJK is subdivided into one of the following: (1) disruption of the posterior osseo-ligamentous complex (ligamentous failure), (2) UIV or UIV + 1 fracture (bony failure), or (3) pull-out of instrumentation (bone-implant interface failure) 14 .PJF was defined as any pain, neurological deficit, compression fracture, or implant failure necessitating revision surgery.

Hounsfield units (HU) measurement
HU were measured from the preoperative and six month-postoperative CT scans at the UIV and UIV-1 in an integrative manner using the Picture Archiving and Communication System (PACs, INFINITT M6, Seoul, Korea) and the Coreline Aview software (v1.1.40,Seoul, Korea) in order to assess the amount of bone formation as a result of rhBMP-2 radiographically [15][16][17] .After obtaining informed consent about CT examination with radiation risk, follow-up CT at 6 months was performed.HU measurement using PACs is conducted according to the method outlined in Schreiber's study.(Fig. 3) 18 .After the CT sagittal view was divided into three sections based on the vertebral body height, HU was calculated by identifying an oval-shaped ROI in the corresponding axial cut that contains only the trabecular bone.The HU of the related level vertebral body was determined using the average value of HU measured in each of the three sections.The procedure of measuring HU using Coreline Aview software was as follows (Fig. 4a and 4b): The region of interest (ROI) was set roughly by manual at the axial, sagittal, and coronal regions.Then the upper and lower limits of HU were set so that only trabecular bone in the vertebral body were included.Then, an upper limit of 1,500 HU and a lower limit of 100 HU were applied using the software to exclude the lung parenchyma, cortical bone of the vertebral body, pedicle screws, and metal artifacts.Finally, additional manual manipulation and ROI confirmation were performed to avoid measurement bias.Then, the software three-dimensionally integrated the HU of the trabecular bones of the UIV or UIV-1.

Patient reported outcome measurements (PROMs)
The Oswestry disability index (ODI), EuroQOL (EQ-5D), and Scoliosis Research Society questionnaire (SRS-22) were used to assess surgical outcomes 19,20 .The ODI is a self-administered questionnaire that measures "back-specific function" on a 10-item scale with six response categories each 20 .The EQ-5D is a 5-dimensional health state classification; the five dimensions are mobility, self-care, usual activities, pain/discomfort, and anxiety/depression 19,21 .EQ-5D "health status was defined by selecting one level from each dimension.The EQ-5D preference-based measure can be regarded as a continuous outcome scored on a 0 to 1.00 scale, with 1.00 indicating "full health" and 0 representing death.These data were collected preoperatively and reassessed three and six months after surgery.

Statistical Analysis
Continuous variables between the groups were compared using an independent t-test.Values are presented as mean ± standard deviation.Categorical variables were compared using the χ 2 test.Given the mean difference in the PJK incidence ratio between the study group and the control group, a post hoc power analysis was performed with an alpha value of 0.05 using G*power 3.1 22 .All statistical analyses were performed using SPSS version 26 software (SPSS Inc., Chicago, IL, USA).Statistical significance was set at p < 0.05.

Patient characteristics
A control group of 75 patients were included at this study.Other demographic data, including height, weight, body mass index, bone mineral density, hand grip strength, past medical histories, and osteoporosis medication, did not differ significantly between the study and control groups (Table 1).

Radiographic parameters and PJK/PJF
Preoperative spinopelvic parameters were not significantly different between the two groups except for lumbar lordosis [-5.3 ± 24.1 vs. 7.1 ± 20.0 for the study and control groups, respectively (p = 0.01)], implying smaller mean lumbar lordosis in the study group (Table 1).The PI-LL mismatch and SVA values were comparable between the two groups during the postoperative phase (Table 2).The incidence of PJK was 2 of 25 (8%) patients in the study group, which was significantly lower than that in the control group, that is, 24 of 75 (32%) patients (p = 0.02).The odds ratio of PJK in the study group was 0.185 (0.04-0.848, 95% CI) (Table 2).One of the two PJK cases in the study group developed bony failure-type PJF, whereas 10 of the 11 PJF patients in the control group (p = 0.29) was bony failure-type.The post hot power analysis confirmed the difference in mean and standard deviation in the PJK incidence ratio between the both groups with an alpha value of 0.05 and a statistical power of 80.0%.

Hounsfield unit measurements
The HU was measured to radiologically confirm new bone formation by the effect of rhBMP-2.While the HU of the UIV at six months after surgery increased significantly compared with that in the preoperative scan (387.2 ± 41.9 vs. 318.8± 44.3, p < 0.001), HU at UIV-1 showed no significant difference (346.4 ± 45.2 vs. 330.8± 48.0, p = 0.250).This resulted in a significantly higher HU at the UIV compared with the UIV-1 in the 6 months' postoperative scan (387.2 ± 41.9 vs. 346.4± 45.2, p = 0.003).Similar findings were observed with the Figure 3. Measurement of Hounsfield Units (HU) from computed tomography using the Scheriber measurement methods.HU was measured within a circular range in the axial view in three sections divided by height.The HU of the vertebral body was defined as the average HU value of the three sections.In the vertebral body where the pedicle screw was inserted, HU was measured in an oval shape in the space between the pedicle screws.Afterwards, the process is the same.Schreiber's HU measurement method.Six months after surgery, there was a statistically significant increase in HU at the UIV level compared to preoperative state.(158.8 ± 36.6 vs. 138.1 ± 40.2, p = 0.001), and there was also a difference in HU between UIV and UIV-1 in the 6 months' postoperative scan (158.8 ± 36.6 vs. 132.7 ± 26.6, p < 0.001).(Table 4).A representative cut of the postoperative CT scan for new bone formation surrounding the UIV pedicle screw where the rhBMP-2 was applied is shown in Fig. 5 and Fig. 6a.This finding is more evident when compared with the control group (Fig. 6b).

Complications
There were no revision cases among the 25 patients in the study group.However, revision surgery which extended posterior fixation and fusion at least 3 upper levels and posterior decompression at PJF level were performed for neurologic deficit with PJK that occurred 6 months after surgery in 5 of the 75 control group patients.Any subchondral sclerosis was not noticed in the study group.

Discussion
This study presents a novel surgical strategy for preventing PJK and PJF after ASD surgery.Transpedicular injection of rhBMP-2 with a β-TCP carrier into the UIV appears to reduce the incidence of PJK by reinforcing trabecular bone formation around the pedicle screws.The enhanced bone quality of the UIV leads to higher resistance to compression fractures and stronger pullout strength of the pedicle screws.The marked decrease in www.nature.com/scientificreports/ the incidence of PJK led to a low occurrence of PJF during the first six months after surgery.The superior PROMs of the study group may have been rooted in the lower PJF rate since other radiographic parameters such as the amount of surgical correction and radiological outcomes were similar between the both groups.Notably, rhBMP-2 promotes osteoinduction and enhances allograft incorporation, increasing the rate of interbody fusion 11 .More recently, the topical placement of rhBMP-2 with a scaffold resulted in bone growth in the field of oral and maxillofacial surgery 12,23 .In an animal study, injection of rhbmp-2 and bone cement into the femoral condyle resulted in bone formation through osteogenesis 24 .Localized bone augmentation through local administration of rhBMP-2 in our study group would likely share a biological mechanism.This strategy is distinct from other PJK prevention strategies, such as cement or ligament augmentation, in that it does not violate natural biology or necessitates additional anatomical exposure.Reducing the rate of PJF and, consequently, the rate of revision surgery in the ASD population would reduce the burden on patients and benefit the healthcare community by reducing the required costs of the process.
New bone formation surrounding the pedicle screw as the result of applying rhBMP-2 can also be used for other surgeries where robust integration between implant and bone is necessary.Many surgeons tried to make a stronger screw fixation strength at osteoporotic vertebra with large diameter and longer pedicle screw, cortical  www.nature.com/scientificreports/bone trajectory, more converge angle, with cementation.The technique of this study is thought to be one way to increase the integration strength between implant and bone [25][26][27][28] .The majority of PJK cases occur early in the postoperative course, reported as 66% within 3 months 14 .Therefore, the fate of the proximal junction of the long-fusion construct was mostly determined during this early period.Meanwhile, the action of rhBMP-2 tracked by HU measurement in the interbody fusion setting showed that bone integration was significantly activated as early as six months postoperatively and reached a plateau at 12 months postoperatively 11 .Hence, patients in the study group were followed up six months after surgery with a CT scan to survey the incidence of PJK and search for any sign of enhanced trabecular bone formation at the UIV.For the quantitative analysis, we measured HU.Measurement of HU in the setting of vertebral body-retaining implants unavoidably encounters the risk of overmeasurement due to metal artifacts.It also depends largely on how the researcher sets the ROI.To overcome this issue, we utilized two different methods.All pedicle screws inserted into the patient's vertebral body were the same product, and the function of the software program was used to minimize metal artifacts.HU, which exceeds the HU of cancellous bone, was set to be automatically excluded.In this study, we set the high cut-off value as 1500 HU to minimize the intervention by metal artifacts.The significant increase in HU at UIV, even compared with UIV-1, may be a sign of new bone formation due to rhBMP-2 injection.
This study had some limitations.First, the sample size was small, and the follow-up period was short.Only 25 patients were injected with rhBMP-2 in the UIV.There were difficulties in deriving the sample size due to the lack of prior research.However, post hoc power analysis confirmed the difference in mean and standard deviation in the PJK incidence ratio between the both groups with an alpha value of 0.05 and a statistical power of 80.0%.Further studies with a larger sample size or randomized controlled trials are necessary to reach a solid conclusion.A longer follow-up would also be beneficial, although it is generally accepted that most PJKs, especially bony failure, occur less than six months after surgery.Second, the ideal dose of rhBMP-2 for this study has not been determined, as this is the first trial focused on the prevention of PJK.After conducting a comprehensive literature review, we have chosen to use 0.5 mg of rhBMP-2 in this preliminary study.This decision is based on the assumption that the required amount of rhBMP-2 may be comparable to or less than that used in lumbar fusion surgery at a single level.Third, β-TCP is a radiopaque material that may remain unabsorbed in postoperative CT scans six months after surgery.However, a recent study utilizing the same combination of rhBMP-2 and β-TCP for alveolar bone augmentation demonstrated increased radiolucency in the radiograph at three months after implantation as compared to the immediate postoperative radiograph 29 .Hence, we do not believe that β-TCP can lead to overestimation when measuring HU.
In conclusion, the intraoperative transpedicular injection of rhBMP-2 with β-TCP carrier into the UIV reduced the incidence of bony failure in the proximal junction, thus necessitating revision surgery.It is easily applicable because the technique does not require a change in the surgical strategy of the surgeon, nor does it require any significant additional surgical procedures.

Figure 4 .
Figure 4. (a) Hounsfield Units (HU) measurement of vertebral body without pedicle screw from computed tomography using the AVIEW software.An upper limit of 1500 HU was used to exclude metal artifacts caused by screws and cortical bone, and a lower limit of 100 HU was used to exclude disc and other soft tissues.(b) Vertebral body with pedicle screw HU measurement.Upper and lower limit of HU get rid of pedicle screw and metal artifacts.

Figure 5 .
Figure 5. Coronal images of three representative patients of the uppermost instrumented vertebra (UIV) rhBMP-2 injection group six month after surgery.Signs of trabecular bone growth are seen around the UIV pedicle screws (arrows).

Figure 6 .
Figure 6.(a) Representative cases, patient who applied rhBMP-2 with β-TCP carrier showed high density at sagittal cut (arrows) of computed tomography at 6 months after surgery.(b) On the other hand, patient who underwent surgery without rhBMP-2 in the control group showed no enhancement around the pedicle screw at uppermost instrumented vertebra (UIV) level.

Table 4 .
Hounsfield unit measurement at the UIV and UIV-1 from the preoperative CT scans and six months postoperative CT scans of the study group.UIV, uppermost instrumented vertebra; CT, computed tomography.p-values < 0.05 are shown in bold.